1. Field of the Invention
The present invention relates to a gel charger for charging a plurality of capillary columns used in a multi-capillary electrophoretic apparatus with a gel serving as a medium for electrophoresing samples.
2. Description of the Prior Art
An electrophoretic apparatus is used for separating and analyzing protein, peptide, sugar or the like, and plays an important part particularly for analysis of the base sequence of DNA.
A DNA sequencer having high sensitivity, a high speed and high throughput is necessary for sequence determination for DNA such as a human genome having long base sequence. As an example, a multi-capillary electrophoretic apparatus formed by arranging a plurality of capillary columns charged with a gel in place of a flat plate type slab gel is proposed. With such capillary columns, samples can not only be readily handled or injected but also electrophoresed at a high speed, and detected in high sensitivity as compared with the slab gel. If a high voltage is applied to the slab gel, a band is spread or a temperature gradient is caused due to influence by Joulean heat However, the capillary columns hardly cause such a problem and can perform detection in high sensitivity with small band spreading even if performing high-speed electrophoresis with application of a high voltage.
Capillary gel electrophoresis using a supporter (separation medium) having a molecular sieving effect in a glass capillary column having an inner diameter of 10 to 200 xcexcm separates nucleic acid or protein by the molecular sieving effect of the separation medium. The separation medium is formed by cross-liked polyacrylamide prepared by polymerizing a cross-linking gel in the capillary column or a previously polymerized high polymer such as linear polyacrylamide or hydroxyethyl cellulose charged into the capillary column.
An initial capillary electrophoretic apparatus employs a single capillary column. In this case, an end of the capillary column is dipped in a gel stored in a container, which in turn is closed and pressurized for forcing and charging the gel into the capillary column. Alternatively, an end of the capillary column is dipped in the gel and another end of the capillary column is decompressed for sucking and charging the gel into the capillary column.
In a multi-capillary electrophoretic apparatus, a plurality of capillary columns are mounted on the multi-capillary electrophoretic apparatus in a state two-dimensionally arranged on a sample injection side and aligned with each other on a plane on a detection side. The plurality of capillary columns are preferably set in a capillary cassette in which the arrangement is fixed by a holder, in consideration of operability.
It is extremely troublesome and impossible in practice to charge the capillary columns one by one with a gel in the state of the capillary cassette. Therefore, it is desired to make it possible to readily charge all capillary columns included in a unit capillary cassette with the gel.
In relation to a separation medium polymerizing a gel in a capillary column, the state of the inner wall surface of the capillary column influences the polymerized state of the gel. If the state of the inner wall surface is inferior, the polymerized state of the gel is deteriorated to increase the frequency of generating bubbles in the capillary column during electrophoresis. Furthermore, if the state of the inner wall surface is inferior, frequency that the gel comes out from an end of the capillary column on a sample injection side is increased whether or not the gel is a cross-linked gel or a high polymer. Thus, reproducibility of an electrophoresis result is disadvantageously deteriorated.
While pretreatment of introducing an acidic solution into the capillary column or the like must be performed for solving this problem, it is extremely troublesome and impossible in practice to pretreat each capillary column contained in a capillary cassette.
Accordingly, an objective of the present invention is to provide a gel charger capable of readily pretreating and charging all capillary columns contained in a capillary cassette with a gel and improving reproducibility of an electrophoresis result in capillary electrophoresis.
The present invention is directed to an apparatus for charging a plurality of capillary columns of a capillary cassette, in which ends of the capillary columns, mounted on a multi-capillary electrophoretic apparatus, on a sample injection side are two-dimensionally arranged through a holding member of a holder and fixed with airtightness between the same and the holding member with a gel.
The capillary columns can be charged with the gel by either suction or pressurization. When charged with the gel, the capillary columns are not sealed one by one and subjected to suction or pressurization, but the holder airtightly fixing the capillary columns seals all capillary columns of the capillary cassette to charge all capillary columns with the gel simultaneously.
A number of silanol groups (xe2x80x94SiOH) are present on inner wall surfaces of glass capillary columns. The states of the silanol groups vary with the capillary columns. The states of the silanol groups remarkably influence a polymerized state of the gel, and hence the difference between the states of the silanol groups varying with the capillary columns vary the polymerized state of the gel.
Therefore, the apparatus according to the present invention introduces acid into the capillary columns before charging the same with the gel or a polymer for arranging the states of the silanol groups on the inner wall surfaces of the capillary columns (hereinafter referred to as acid treatment) and bringing the silanol groups into states suitable for gel polymerization. Consequently, the polymerized state of the gel in the capillary columns can be stabilized. On the other hand, acid inhibits gel polymerization and hence the capillary columns subjected to acid treatment are thereafter washed with pure water. If moisture remains in the capillary columns, gel polymerization is inhibited or the gel concentration is reduced. As a result, the capillary columns are dried with inert gas.
In a system performing gel charging by suction, a gel charger according to the present invention comprises a chamber airtightly fixing ends of capillary columns on a sample injection side, an inert gas supply mechanism supplying inert gas, decompression means decompressing the chamber, and an introduced substance selection mechanism for supplying ends of the capillary columns on a detection side opposite to the sample injection side with inert gas, an acidic solution, pure water or a gel. The chamber is provided on its upper surface with an opening, which in turn is provided with closure means receiving the ends of the capillary columns on the sample injection side therein and closing the opening with a holder, and a gas outlet is further provided. The decompression means is provided on the gas outlet of the chamber. The introduced substance selection mechanism comprises an inert gas supply port supplied with inert gas from the inert gas supply mechanism, an acidic solution container storing an acidic solution, a pure water container storing pure water and a gel container storing a gel, and comprises a mechanism moving the inert gas supply port, the acidic solution container, the pure water container and the gel container as well as ends of the capillary columns on a detection side opposite to the sample injection side so that the ends of the capillary columns on the detection side are inserted in any of the inert gas supply port, the acidic solution container, the pure water container and the gel container.
In operation, the gel charger closes the opening of the chamber with the closure means through the holder of the capillary cassette thereby fixing the detection side of the capillary cassette to the introduced substance selection mechanism. Then the gel charger arranges the acidic solution container, the pure water container, the gel container and the inert gas supply port on prescribed positions of the introduced substance selection mechanism.
First, the introduced substance selection mechanism dips forward ends of all capillary columns on the detection side of the capillary cassette in the acidic solution stored in the acidic solution container while the decompression means decompresses the chamber for inhaling the acidic solution into the capillary columns and performing acid treatment on the inner wall surfaces of the capillary columns.
Secondly, the introduced substance selection mechanism dips the forward ends of all capillary columns on the detection side of the capillary cassette in the pure water stored in the pure water container while the decompression means decompresses the chamber for inhaling the pure water into the capillary columns and discharging the acidic solution from the capillary columns.
Thirdly, the introduced substance selection mechanism inserts the forward ends of all capillary columns on the detection side of the capillary cassette into the inert gas supply port for supplying the inert gas from the inert gas supply mechanism and the decompression means decompresses the chamber for inhaling the inert gas into the capillary columns and drying the capillary columns.
Lastly, the introduced substance selection mechanism dips the forward ends of all capillary columns on the detection side of the capillary cassette in the gel stored in the gel container while the decompression means decompresses the chamber for inhaling the gel into the capillary columns. Thus, gel charging is completed.
In a system performing gel charging by pressurization, a gel charger according to the present invention comprises a chamber fixing ends of capillary columns on a sample injection side while keeping airtightness and storing a gel therein, a gel container, storing the gel, detachably arranged in the chamber, pressurization/decompression means pressurizing or decompressing the chamber, an inert gas supply mechanism supplying inert gas, and an introduced substance selection mechanism for supplying ends of the capillary columns on a detection side opposite to the sample injection side with inert gas, an acidic solution or pure water or connecting the same to a drain. The chamber is provided on its upper surface with an opening, which in turn is provided with closure means receiving the ends of the capillary columns on the sample injection side therein and closing the opening with a holder, and a gas outlet is further provided. The pressurization/decompression means is provided on the gas outlet of the chamber. The gel container is so arranged in the chamber that the gel comes to a position where the ends of the capillary columns on the sample injection side are dipped while the opening of the chamber is closed with the holder. The introduced substance selection mechanism comprises an inert gas supply port supplied with inert gas from the inert gas supply mechanism, an acidic solution container storing an acidic solution, a pure water container storing pure water and a drain container, and comprises a mechanism moving the inert gas supply port, the acidic solution container, the pure water container and the drain container as well as the ends of the capillary columns on the detection side so that ends of the capillary columns on a detection side opposite to the sample injection side are inserted in any of the inert gas supply port, the acidic solution container, the pure water container and the drain container.
In operation, the gel charger of this system closes the opening of the chamber with the closure means through a holder of a capillary cassette while not arranging the gel container in the chamber. The gel charger fixes a detection side of the capillary cassette to the introduced substance selection mechanism, and arranges the acidic solution container, the pure water container, the inert gas supply port and the drain container on prescribed positions of the introduced substance selection mechanism.
First, the introduced substance selection mechanism dips forward ends of all capillary columns on the detection side of the capillary cassette in the acidic solution stored in the acidic solution container while the pressurization/decompression mechanism decompresses the chamber for inhaling the acidic solution into the capillary columns and performing acid treatment on inner wall surfaces of the capillary columns.
Secondly, the introduced substance selection mechanism dips the forward ends of all capillary columns on the detection side of the capillary cassette in the pure water stored in the pure water container while the pressurization/decompression mechanism decompresses the chamber for inhaling the pure water into the capillary columns and discharging the acidic solution from the capillary columns.
Thirdly, the introduced substance selection mechanism inserts the forward ends of all capillary columns on the detection side of the capillary cassette into the inert gas supply port and supplies the inert gas from the inert gas supply mechanism, while the pressurization/decompression mechanism decompresses the chamber for inhaling the inert gas into the capillary columns and drying the capillary columns.
Lastly, the holder is temporarily detached from the opening of the chamber, the gel container is arranged in the chamber and thereafter the opening of the chamber is closed with the holder again. Thereafter the introduced substance selection mechanism inserts the forward ends of all capillary columns on the detection side of the capillary cassette in the drain container while the pressurization/decompression means pressurizes the chamber for injecting the gel into the capillary columns. The drain container receives an excess amount of gel overflowing the ends of the capillary columns on the detection side.
When a plurality of columns forming a capillary array mounted on a multi-capillary electrophoretic apparatus are charged with a gel solution, the respective capillary columns are not directly closed and fixed but sealed with the closure means and charged with the gel through a sample injection side cassette holder airtightly fixing the plurality of capillary columns, whereby the plurality of capillary columns can be simultaneously charged with the gel. Simplicity of mounting and airtightness in mounting can be compatibly attained.
The plurality of capillary columns can be simultaneously charged with the gel solution, whereby the maximum merit of improving the throughput by simultaneously electrophoresing a plurality of samples in multi-capillary electrophoresis is not damaged by a process of gel charge serving as pretreatment of the electrophoresis
Furthermore, the acidic solution arranges states of silanol groups on the inner wall surfaces of the capillary columns, whereby the states of the inner wall surfaces of the capillary columns can be optimized in relation to gel charging and gel polymerization. Consequently, a polymerized state in gel polymerization is improved, a bubble generation ratio in application of an electrophoresis voltage is reduced and displacement of the gel is reduced, whereby reproducibility of a electrophoresis result can be improved.
The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings.